A method for detecting human soluble asialoglycoprotein receptor

A sialic acid glycoprotein, soluble technology, applied in the field of immunological detection, can solve the problems of high preparation cost, inaccurate results, time-consuming and laborious, etc., and achieves the effects of good repeatability, high practicability and good repeatability

Active Publication Date: 2021-03-26
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The double-antibody sandwich method kit needs to use two different antibodies against different sites of ASGPR. The antibody preparation process is time-consuming and labor-intensive, and the preparation cost is relatively high, resulting in expensive kits and high detection costs for users; in addition, The detection limits of some existing ELISA kits are not suitable for the detection of sASGPR in human serum, which makes the detection of serum samples inconvenient and the results are not accurate enough

Method used

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  • A method for detecting human soluble asialoglycoprotein receptor
  • A method for detecting human soluble asialoglycoprotein receptor
  • A method for detecting human soluble asialoglycoprotein receptor

Examples

Experimental program
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Embodiment 1

[0039] The preparation of embodiment 1 ligand GSA

[0040] Add 20mg human serum albumin to a clean reaction bottle, then add 20mL 0.5M MES biological buffer (pH5.25) to fully dissolve human serum albumin, then add 108.6mg galactosamine and 62mg EDC to fully dissolve, add After all the samples were completed, the reaction bottle was placed in a 37°C oil bath and reacted for 16 hours. After the reaction, add 3.4 mL of 1M CH 3 COOH solution (pH 4.5) to terminate the reaction. After the reaction was terminated, the reaction solution was transferred to In the MLtra centrifugal filter, centrifuge at 5000×g for 20min, pour off the filtrate in the lower centrifuge tube, and add 10mM CH to the upper filter 3 The COOH solution (pH 7) was restored to the original volume of the sample, centrifuged again at 5000×g, and ultrafiltration was repeated several times to remove unreacted galactosamine and reduce the concentration of sodium acetate. The final GSA solution obtained after sever...

Embodiment 2A

[0041] The purification of embodiment 2ASGPR standard substance

[0042] Take 150mL of the HepG2 cell supernatant collected in advance and centrifuge for 5min (3500×g) in a centrifuge, take the centrifuged supernatant and add it to a 50mL Concentrate in a MLtra centrifugal filter (3500×g) to a final volume of 8 mL. Add 8 mL of lysis buffer to the concentrated HepG2 cell supernatant, mix well, and lyse at 4°C for 2-3 hours. After the lysis is completed, centrifuge at 20000×g for 30min, take the supernatant into a new centrifuge tube, add 800μL 1M CaCl 2 , and incubated on ice for 30min. Centrifuge again at 20,000×g for 30 min, and discard the precipitate. The lactose-agarose bead column was pre-equilibrated with 50 mL of washing buffer I, the lactose-agarose beads were fully transferred to a centrifuge tube, and combined with the centrifuged cell supernatant overnight at 4°C. Fully transfer the bound lactose-agarose beads to the column, and wash the column with 10 mL of wa...

Embodiment 3

[0043] Example 3 Selection of Primary Antibody

[0044] Purchase two primary antibodies of ASGPR1 mouse origin: ASGPR1 / 2 (E-1) (Santa Cruz Biotechnology, sc-166633) and ASGPR1 (A-5) (Santa Cruz Biotechnology, sc-393849), respectively referred to below Mouse anti-human ASGPR-1 monoclonal antibody, mouse anti-human ASGPR-2 monoclonal antibody. Coat two 96-well ELISA plates with 10 μg / mL GSA, add 100 μL GSA to each well, and coat at 4°C for 24 hours. After coating, wash 5 times with PBST for 3 minutes each time, and pat dry. Add 300 μL of 1% BSA to each well to block the whole well, and block overnight at 4°C. After blocking, wash 3 times with PBST for 3 min each time, and pat dry. Set ASGPR diluent as negative control well, quality control well and 0.15 μg / mL, 0.50 μg / mL, 2.00 μg / mL, 8.00 μg / mL ASGPR standard as sample wells respectively, add negative control and samples in turn to enzyme In the target plate, 4 replicate wells were made for the sample, and the sample volume in...

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Abstract

An ELISA method for detecting human soluble asialoglycoprotein receptor (sASGPR). The method is based on a specific ligand of ASGPR and the specific recognition of ASGPR. Galactosylated serum albumin (GSA) is selected as the specific ligand of ASGPR, and the GSA is prepared from human serum albumin and has the advantages of being low in cost, easy to prepare, easy to preserve and the like. The detection limit of the method is applicable to detection of sASGPR in a human serum sample, there is no need to use special and large instruments and equipment, and the method can be carried out in an ordinary laboratory, has the advantages of being high in specificity, good in stability, easy and convenient to operate, low in cost and the like, and provides certain reference value for clinical liver function assessment.

Description

technical field [0001] The invention relates to a method for detecting human body's soluble asialoglycoprotein receptor, which belongs to the field of immunological detection. Background technique [0002] For decades, liver disease has been an important disease with a high incidence worldwide. Liver damage such as cirrhosis, hepatitis, and fatty liver is the only way for liver cancer. Liver damage seriously threatens the health of people in my country and the world and life. Currently, the main detection methods for liver injury include histopathological diagnosis, serological diagnosis and imaging diagnosis. Among them, the main clinical serum markers for serological diagnosis are alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and serum bilirubin (BIL), etc., but these indicators have low specificity. The shortcomings of these indicators, in addition to liver damage will have an impact on these indicators, some other diseases ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 胡静尹健施奇敏
Owner JIANGNAN UNIV
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